Quantitative determination of tolbutamide and its metabolites in human plasma and urine by high-performance liquid chromatography and UV detection

An isocratic, high-performance liquid chromatography method has been developed for simultaneous determination of the oral antidiabetic tolbutamide and two of its metabolites, 4-hydroxytolbutamide and carboxytolbutamide, in human plasma and urine. The method was based on simple one-step liquid-liquid extraction with tertiary-butyl methyl ether as extraction solvent. The chromatographic eluent was 23:77 (v/v) methanol: 0.01 M aqueous sodium acetate buffer pH 3.0, and the UV detection was performed at a wavelength of 230 nm. The limit of detection was 0.1 mu M for tolbutamide in plasma and 1.5 mu M, 0.5 mu M, and 0.75 mu M for carboxytolbutamide, 4-hydroxytolbutamide, and tolbutamide, respectively, in urine. The limit of quantitation was 0.5 mu M for tolbutamide in plasma and 2 mu M, 0.75 mu M, and 1.25 mu M for carboxytolbutamide, 4-hydroxytolbutamide, and tolbutamide, respectively, in urine. The overall mean recoveries ranged from 91% to 109% for tolbutamide in plasma and from 80% to 98% in urine for all three compounds.